Detection Of Anti-TMV Activity Of Brucein D By Green Fluorescent Protein Labeling

Tobacco mosaic virus(TMV),one of the most serious virus which infect tobacco plants,is widely distributed and have caused enormous economic losses. To prevent this dissease, the plant originated active substances are effective and environment friendly choices.Therefore, selecting an effective anti-virus inhibitor from plant extractions is very important. An active substance called Brucein D has been isolated from the herb species Brucea javanica(L.)Merr in our lab. Preliminary observations showed that it could significantly inhibit the replication of TMV.Using green fluorescent protein (GFP) as reporter to observe the expression of TMV in plants would be more direct and convenient. In this study, GFP gene was inserted into the TMV genome to construct the plasmid p35S-30B::GFP. Then we used this TMV-based expression vector to investigate the effects of Brucein D on the multiplication and movement of TMV in plant and in protoplast as well.We infiltrated p35S-30B::GFP harbored in Agrobacterium strain EHA 105 into leaves of Nicotiana benthamiana plants using a needle-less syring. Before infiltration, different concentrations of Brucein D were applied. Based on the observation of the expression of the GFP after a period of time, we concluded that 10μg/mL is the best working concentration for Brucein D; To test this result more convincingly, PCR and western blot experiments were conducted to detect the expression of CP and GFP in newly developed leaves. The results indicated that: CP and GFP can be detected in plants inoculated with TMV particles in the absence 10μg/ml Brucein D; CP and GFP can not be detected in plants inoculated with TMV particles plus 10μg/ml Brucein D. Similarly, GFP fluorescence can be observed in systemic leaves of plants inoculated with p35S-30B::GFP alone, but can not be in systemic leaves of plants inoculated p35S-30B::GFP plus 10μg/mL Brucein D. The results of the experiments also showed that the inhibitory effects of Brucein D on TMV were time-dependent. Observation of the expression of GFP confirmed that the inhibitory effects of Brucein D on TMV could persist for 3-4weeks.Nicotiana benthamiana mesophyll protoplasts were obtained. Factors affacting the production of mesophyll protoplasts were investigated during ours research. The results showed that the optimum conditions for isolating Nicotiana benthamiana mesophyll protoplasts were: 4 hours of enzyme digestion, using newly developed leaves, 30 min of vacuum filtrating, the enzyme solution pH 6.0. p35S:30B:GFP was successfully delivered into the protoplasts by using PEG-mediated transformation. Expression of GFP was observed 48 hours after transformation.The effects of Brucein D on TMV was then assessed 48 hours after PEG-mediated delivery of the p35S:30B:GFP into protoplasts derived from Nicotiana benthamiana leaves. The results showed that the Brucein D, at the concentration 10μg/mL, could also inhibit multiplication of TMV in isolated cells.In this study, tobacco plants K326 were inoculated with TMV particles, wich were mixed with Brucein D at 10μg/mL. ELISA analysis showed that Brucein D could also effectly inhibit TMV replication in K326 plants.